Methods of Increasing the Cellulolytic Enhancing Activity of a Polypeptide

ABSTRACT

The present invention relates to methods of increasing the activity of a GH61 polypeptide having cellulolytic enhancing activity, comprising: adding a divalent copper cation to a composition comprising the GH61 polypeptide having cellulolytic enhancing activity, wherein the presence of the divalent copper cation and the GH61 polypeptide having cellulolytic enhancing activity increases degradation or conversion of a cellulosic material by an enzyme composition compared to the GH61 polypeptide having cellulolytic enhancing activity without the divalent copper cation. The present invention also relates to compositions, methods for degrading or converting a cellulosic material, and methods for producing a fermentation product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/004,102, filed Dec. 20, 2013, which is a 35 U.S.C. §371 nationalapplication of PCT/US2012/028594 filed Mar. 9, 2012, which claimspriority or the benefit under 35 U.S.C. §119 of U.S. ProvisionalApplication Ser. No. 61/481,534, filed May 2, 2011, and U.S. ProvisionalApplication Ser. No. 61/451,055, filed Mar. 9, 2011, the contents ofwhich are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compositions for increasingand stabilizing the activity of a GH61 polypeptide having cellulolyticenhancing activity.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

WO 2005/074647, WO 2008/148131, and WO 2011/035027 disclose isolatedGH61 polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascusaurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864,and WO 2009/085868 disclose isolated GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromMyceliophthora thermophila. WO 2010/138754 discloses isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Aspergillus fumigatus. WO 2011/005867discloses isolated GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Penicillium pinophilum. WO2011/039319 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascus sp.WO 2011/041397 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Penicillium sp.(emersonii). WO 2011/041504 discloses isolated GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromThermoascus crustaceus. WO 2008/151043 discloses methods of increasingthe activity of a GH61 polypeptide having cellulolytic enhancingactivity by adding a soluble activating divalent metal cation to acomposition comprising the polypeptide.

It would be an advantage in the art to improve the activity andstability of GH61 polypeptides having cellulolytic enhancing activity.

The present invention relates to methods and compositions for increasingand stabilizing the activity of GH61 polypeptides having cellulolyticenhancing activity.

SUMMARY OF THE INVENTION

The present invention relates to methods of increasing the activity of aGH61 polypeptide having cellulolytic enhancing activity, comprising:adding a divalent copper cation to a composition comprising the GH61polypeptide having cellulolytic enhancing activity, wherein the divalentcopper cation is present at a concentration of about 0.0001 mM to about20 mM during degradation or conversion of a cellulosic material.

The present invention also relates to methods of increasing thestability of a GH61 polypeptide having cellulolytic enhancing activity,comprising: adding a divalent copper cation to a composition comprisingthe GH61 polypeptide, wherein the divalent copper cation is present at aconcentration of 0.0001 mM to about 20 mM.

The present invention also relates to methods of increasing the activityand the stability of a GH61 polypeptide having cellulolytic enhancingactivity, comprising: adding a divalent copper cation to a compositioncomprising the GH61 polypeptide, wherein the divalent copper cation ispresent at a concentration of 0.0001 mM to about 20 mM.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition comprising a GH61 polypeptide havingcellulolytic enhancing activity and a divalent copper cation, whereinthe divalent copper cation is present at a concentration of about 0.0001mM to about 20 mM.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising a GH61 polypeptide havingcellulolytic enhancing activity and a divalent copper cation, whereinthe divalent copper cation is present at a concentration of about 0.0001mM to about 20 mM; (b) fermenting the saccharified cellulosic materialwith one or more (e.g., several) fermenting microorganisms to producethe fermentation product; and (c) recovering the fermentation productfrom the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g. several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition comprising a GH61polypeptide having cellulolytic enhancing activity and a divalent coppercation, wherein the divalent copper cation is present at a concentrationof about 0.0001 mM to about 20 mM.

The present invention also relates to compositions comprising a GH61polypeptide having cellulolytic enhancing activity and a divalent coppercation, wherein the divalent copper cation is present at a concentrationof about 0.0001 mM to about 20 mM during degradation or saccharificationof a cellulosic material and the presence of the divalent copper cationand the GH61 polypeptide increases the degradation or conversion of thecellulosic material by an enzyme composition compared to the GH61polypeptide without the divalent copper cation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an EPR spectrum (140 K) of 1.0 mM copper(II) nitrate in 10mM sodium acetate buffer.

FIG. 2 shows EPR spectra (150 K) of demetallated Thermoascus aurantiacusGH61A polypeptide and Thielavia terrestris GH61E polypeptide.

FIGS. 3A and 3B show (A) an EPR spectrum (140 K) of copper(II)-T.aurantiacus GH61A polypeptide in 10-20% glycerol and 1 mM sodiumacetate; and (B) of copper(II)-T. terrestris GH61E polypeptide in 10-20%glycerol and 1 mM sodium acetate.

FIGS. 4A and 4B show (A) an EPR spectrum (150 K) of Cu-T. aurantiacusGH61A polypeptide following treatment with excess ascorbate andcellulosic substrate, and (B) a blow-up of the organic radical region.

FIG. 5 shows (1) the effect of 1 μM cupric (copper(II)) ion onhydrolysis of AVICEL® by a Trichoderma reesei cellulase composition inthe absence of a GH61 polypeptide (Cupric ion effect_((no GH61)), whitebars), (2) the effect of cupric ion on hydrolysis of AVICEL® by a T.reesei cellulase composition in the presence of a GH61 polypeptide(Cupric ion effect_((+GH61)), grey bars), and (3) the effect of a GH61polypeptide on hydrolysis of AVICEL® by a T. reesei cellulasecomposition in the presence of cupric ion (GH61 effect, black bars) for1, 3, and 7 days, in the presence of dehydroascorbic acid.

FIG. 6 shows the effect of a GH61 polypeptide on hydrolysis of AVICEL®by a T. reesei cellulase composition in the presence of cupric ion (GH61effect, black bars) and dehydroascorbic acid for 1 day of hydrolysis.

FIG. 7 shows (1) the effect of 100 μM cupric ion on hydrolysis ofAVICEL® by a T. reesei cellulase composition in the absence of a GH61polypeptide (Cupric ion effect_((no GH61)), white bars), (2) the effectof cupric ion on hydrolysis of AVICEL® by a T. reesei cellulasecomposition in the presence of a GH61 polypeptide (Cupric ioneffect_((+GH61)), grey bars), and (3) the effect of a GH61 polypeptideon hydrolysis of AVICEL® by a T. reesei cellulase composition in thepresence of cupric ion (GH61 effect, black bars) for 1, 3, and 7 days,in the absence of dehydroascorbic acid.

FIG. 8 shows the effect of a GH61 polypeptide on hydrolysis of AVICEL®by a T. reesei cellulase composition in the presence of cupric ion (GH61effect, black bars) and absence of dehydroascorbic acid for 7 days ofhydrolysis.

FIGS. 9A and 9B show the thermal stability of the Thermoascusaurantiacus GH61A polypeptide (Ta61A) in the presence of calciumchloride or copper sulfate.

FIG. 10 shows the effect of cupric ion addition on the thermal stabilityof the Thielavia terrestris GH61E polypeptide in the presence andabsence of DTPA chelator.

FIG. 11 shows the effect of 100 μM cupric ion addition on the thermalstability of the Thielavia terrestris GH61E polypeptide at pH 5, 7, and9.

FIG. 12 shows the effect of 100 μM cupric ion addition on the thermalstability of the Aspergillus fumigatus GH61 B polypeptide in thepresence of 1 mM DTPA chelator at pH 5, 7, and 9.

FIG. 13 shows the effect of 100 μM cupric ion addition on the thermalstability of the Penicillium sp. (emersonii) GH61A polypeptide in thepresence of 1 mM DTPA chelator at pH 5, 7, and 9.

FIG. 14 shows the effect of 100 μM cupric ion addition on the thermalstability of the Thermoascus crustaceus GH61A polypeptide in thepresence of 1 mM DTPA chelator at pH 5, 7, and 9.

FIG. 15 shows the effect of 100 μM cupric ion addition on the thermalstability of the Thermoascus aurantiacus GH61A polypeptide in thepresence of 1 mM DTPA chelator at pH 5, 7, and 9.

FIG. 16 shows the effect of CuSO₄ on the transformation of methyleneblue in the presence of the Thermoascus aurantiacus GH61A polypeptideand pyrogallol.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain (Teeri, 1997, Crystalline cellulosedegradation: New insight into the function of cellobiohydrolases, Trendsin Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reeseicellobiohydrolases: why so efficient on crystalline cellulose?, Biochem.Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determinedaccording to the procedures described by Lever et al., 1972, Anal.Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288;and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the presentinvention, the Tomme et al. method can be used to determinecellobiohydrolase activity.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material(including energy crops), agricultural residue, wood (including forestryresidue), municipal solid waste, waste paper, and pulp and paper millresidue (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is herbaceous material (includingenergy crops). In another aspect, the cellulosic material isagricultural residue. In another aspect, the cellulosic material is wood(including forestry residue). In another aspect, the cellulosic materialis municipal solid waste. In another aspect, the cellulosic material iswaste paper. In another aspect, the cellulosic material is pulp andpaper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is wheat straw. In another aspect, thecellulosic material is bagasse. In another aspect, the cellulosicmaterial is corn cob. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is corn fiber.In another aspect, the cellulosic material is rice straw. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is poplar. In another aspect, the cellulosic material is pine.In another aspect, the cellulosic material is willow. In another aspect,the cellulosic material is eucalyptus.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae; submerged plants; emergent plants; and floating-leaf plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has activity asthe mature polypeptide thereof.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE families. Somefamilies, with an overall similar fold, can be further grouped intoclans, marked alphabetically (e.g., GH-A). A most informative andupdated classification of these and other carbohydrate active enzymes isavailable in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitabletemperature, e.g., 50° C., 55° C., or 60° C., and pH, e.g., 5.0 or 5.5.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Increased thermal stability: The term “increased thermal stability”means a higher retention of cellulolytic enhancing activity of a GH61polypeptide in the presence of divalent copper cation compared to theabsence of the divalent copper cation after a period of incubation at atemperature. The increased thermal stability of a GH61 polypeptide canbe assessed, for example, under conditions of one or more (e.g.,several) temperatures. For example, the one or more (e.g., several)temperatures can be any temperature or temperatures in the range of 45°C. to 95° C., e.g., 45, 50, 55, 60, 65, 70, 75, 80, 85, or 95° C. (or inbetween, e.g., 62° C., 68° C., etc.) at one or more (e.g., several) pHsin the range of 3 to 9, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, or 9.0 (or in between) for a suitable period ofincubation, e.g., 1 minute, 5 minutes, 10 minutes, 15 minutes, 30minutes, 45 minutes, or 60 minutes, such that the GH61 polypeptideretains residual activity. However, longer periods of incubation canalso be used.

The increased thermal stability of a GH61 polypeptide can be determinedby differential scanning calorimetry (DSC) using methods standard in theart (see, for example, Sturtevant, 1987, Annual Review of PhysicalChemistry 38: 463-488). The thermal stability of a GH61 polypeptide canalso be determined using any enzyme assay known in the art for GH61polypeptides having cellulolytic enhancing activity. See for example, WO2005/074647, WO 2008/148131 WO 2005/074656, WO 2010/065830, WO2007/089290, WO 2009/085935, WO 2009/085859, WO 2009/085864, WO2009/085868, and WO 2008/151043, which are incorporated herein byreference. Alternatively, the increased thermal stability of a GH61polypeptide can be determined using any application assay where theperformance of the GH61 polypeptide in the presence and absence ofdivalent copper cation are compared. For example, the application assaydescribed in the Examples herein can be used.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. It is also known in the art thatdifferent host cells process polypeptides differently, and thus, onehost cell expressing a polynucleotide may produce a different maturepolypeptide (e.g., having a different C-terminal and/or N-terminal aminoacid) as compared to another host cell expressing the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving enzyme activity.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., andpH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In a preferred aspect, amixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsvrd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”. For purposes of the present invention, the sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes apolypeptide fragment having activity as the mature polypeptide thereof.

Variant: The term “variant” means a polypeptide having enzyme activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of increasing the activity of aGH61 polypeptide having cellulolytic enhancing activity, comprising:adding a divalent copper cation to a composition comprising the GH61polypeptide, wherein the divalent copper cation is present at aconcentration of about 0.0001 mM to about 20 mM during degradation orconversion of a cellulosic material.

The present invention also relates to methods of increasing thestability of a GH61 polypeptide having cellulolytic enhancing activity,comprising: adding a divalent copper cation to a composition comprisingthe GH61 polypeptide, wherein the divalent copper cation is present at aconcentration of 0.0001 mM to about 20 mM.

The present invention also relates to methods of increasing the activityand the stability of a GH61 polypeptide having cellulolytic enhancingactivity, comprising: adding a divalent copper cation to a compositioncomprising the GH61 polypeptide, wherein the divalent copper cation ispresent at a concentration of 0.0001 mM to about 20 mM.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition comprising a GH61 polypeptide havingcellulolytic enhancing activity and a divalent copper cation, whereinthe divalent copper cation is present at a concentration of about 0.0001mM to about 20 mM.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising a GH61 polypeptide havingcellulolytic enhancing activity and a divalent copper cation, whereinthe divalent copper cation is present at a concentration of about 0.0001mM to about 20 mM; (b) fermenting the saccharified cellulosic materialwith one or more (e.g., several) fermenting microorganisms to producethe fermentation product; and (c) recovering the fermentation productfrom the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g. several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition comprising a GH61polypeptide having cellulolytic enhancing activity and a divalent coppercation, wherein the divalent copper cation is present at a concentrationof about 0.0001 mM to about 20 mM.

The presence of the divalent copper cation and the GH61 polypeptidehaving cellulolytic enhancing activity increases the degradation,conversion, or saccharification of the cellulosic material by the enzymecomposition compared to the GH61 polypeptide having cellulolyticenhancing activity without the divalent copper cation.

The present invention also relates to compositions comprising a GH61polypeptide having cellulolytic enhancing activity and a divalent coppercation, wherein the divalent copper cation is present at a concentrationof about 0.0001 mM to about 20 mM during degradation or saccharificationof a cellulosic material and the presence of the divalent copper cationand the GH61 polypeptide increases the degradation or conversion of thecellulosic material by an enzyme composition compared to the GH61polypeptide without the divalent copper cation. In one aspect, thecomposition further comprises one or more (e.g., several) enzymesselected from the group consisting of a cellulase, a GH61 polypeptidehaving cellulolytic enhancing activity, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

Divalent Copper Cations

A divalent copper cation is preferably present at a concentration so theGH61 polypeptide is fully complexed with the divalent copper cation. Theterm “fully complexed with divalent copper cation” means the GH61polypeptide is preferably at least 75%, more preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, or even most preferably 100% in the coppercontaining form, i.e., the GH61 polypeptide is preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, or even mostpreferably 100% active. Complexation of the GH61 polypeptide withdivalent copper cation can be determined using techniques well known inthe art, e.g., electron paramagnetic resonance (EPR).

A GH61 polypeptide fully complexed with divalent copper cation may beobtained during production of the polypeptide by fermentation byincluding a sufficient amount of a divalent copper cation in thefermentation medium. Alternatively, a GH61 polypeptide could bedemetallated by standard methods known in the art, e.g., elution from ametal-chelating resin, or preincubation with metal chelators followed bymembrane or gel filtration to remove the chelators and chelated metal,and replaced with divalent copper cation.

The divalent copper cation concentration yielding optimal GH61 activityon a given pretreated lignocellulosic material can be identified byvarying the concentration of the divalent copper cation to determine theminimum concentration of copper yielding the maximum conversion toglucose. In one aspect, divalent copper cation is added at aconcentration of about 0.0001 mM to about 20 mM, e.g., about 0.0005 mMto about 15 mM, about 0.001 mM to about 10 mM, about 0.005 mM to about 5mM, about 0.01 mM to about 2.5 mM, about 0.05 mM to about 1 mM, or about0.1 mM to about 1 mM to the enzymatic degradation reaction. The properamount of divalent copper cation to insure full complexation of the GH61polypeptide with the divalent copper cation can be determined by EPR.The divalent copper cation is preferably added as a soluble salt, forexample, a chlorate, chloride, chromate, citrate, fluoride, formate,iodide, nitrate, oxalate, perchlorate, selenate, or sulfate salt, or asan insoluble salt, for example, a carbonate, hydroxide, oxide,phosphate, pyrophosphate, or sulfide salt. In one aspect, the solublesalt is CuSO₄ or Cu(NO₃)₂. The divalent copper cation may also bederived from added monovalent copper cation (cuprous) salt, for example,a bromide, chloride, cyanide, fluoride, hydroxide, iodide, or sulfidesalt, or added zero-valent (atomic) metallic copper or copper-containingalloy, when the cation or atom is transformed into divalent coppercation in situ.

It is well known in the art that cellulosic biomass can comprise anumber of divalent metal cations. See, for example, F. B. Salisbury andC. W. Ross: Plant Physiology, Wadsworths Publishing Company, Belmont,Calif. (1992). The cellulosic biomass may be, therefore, in part orwholly, a source of the divalent copper cations. The activating divalentcopper cations may be soluble or insoluble. The term “activatingdivalent copper cation” is defined herein as a divalent copper cationthat is available in solution to increase the activity of a GH61polypeptide having cellulolytic enhancing activity. However, thedivalent copper cations may be unavailable in solution to increase theactivity of a GH61 polypeptide having cellulolytic enhancing activitybecause, for example, they are complexed with a component of thecellulosic biomass, for example, pyrophosphate.

The cellulosic biomass can also provide soluble divalent metal cationsat concentrations that inhibit cellulolysis (hereinafter “inhibitorydivalent metal cation”). For example, an inhibitory divalent metalcation is Zn⁺⁺ when present at mM or higher concentrations.Consequently, under conditions where a mixture of divalent coppercations and inhibitory divalent metal cations are present, an excess ofthe divalent copper cation may be needed to overcome the inhibitoryeffect of the inhibitory divalent metal cations. In such a situation toprevent inhibitory divalent metal cations from adversely affecting theGH61 polypeptide having cellulolytic enhancing activity, the methods ofthe present invention further comprise supplementing the concentrationof the divalent copper cation to insure full complexation of the GH61polypeptide with divalent copper cation. The effective concentration ofthe supplemented divalent copper cation may be in the range of about0.0001 mM to about 20 mM, e.g., about 0.0005 mM to about 15 mM, about0.001 mM to about 10 mM, about 0.005 mM to about 5 mM, about 0.01 mM toabout 2.5 mM, about 0.05 mM to about 1 mM, or about 0.1 mM to about 1mM.

The concentration of divalent cations in cellulosic biomass can bedetermined using any method known in the art, such as atomic absorption,electrochemical electrodes, metal ion biosensors, optical sensors, ortitration by chelation (see, for example, Methods in Enzymology, v. 158(multiple chapters), Haugland, R. P. Handbook of Fluorescent Probes andResearch Chemicals, 6th ed.; Molecular Probes, Inc.: Eugene, Oreg.,1996., Thompson et al. Anal. Chem., 70 (22), 4717-4723, 1998,Inductively Coupled Plasma Mass Spectrometry, Akbar Montaser (Editor)May 1998).

In one aspect, the methods of the present invention further compriseadding a chelator during the degradation or saccharification of thecellulosic material by an enzyme composition. The chelator may be addedso the activity of a GH61 polypeptide is optimal on a given pretreatedlignocellulosic material. In one aspect, the chelator is selected fromthe group consisting of EDTA (ethylenediaminetetraacetic acid), EGTA(ethyleneglycol-bis-(2-aminoethyl)-N,N,N′,N′-tetraacetic acid), DDTA(3,6-dioxaoctamethylenedinitrilotetraacetic acid), EDDS(ethylenediamine-N,N′-disuccinic acid), BAPTA(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), and BIPY(2,2′-bipyridine). In another aspect, the chelator is EDTA. In anotheraspect, the chelator is EGTA. In another aspect, the chelator is DDTA.In another aspect, the chelator is EDDS. In another aspect, the chelatoris BAPTA. In another aspect, the chelator is BIPY. However, any suitablechelator may be used. The effective concentration of the chelator may bein the range of about 0.0001 mM to about 100 mM, e.g., about 0.0002 mMto about 75 mM, about 0.0003 mM to about 50 mM, about 0.0005 mM to about25 mM, about 0.001 mM to about 10 mM, about 0.005 mM to about 5 mM,about 0.01 mM to about 2.5 mM, about 0.05 mM to about 1 mM, or about 0.1mM to about 1 mM. In a preferred aspect, the chelator is added after theGH61 polypeptide is fully complexed with divalent copper cation.

In one aspect, a divalent copper cation increases the activity of a GH61polypeptide having cellulolytic enhancing activity preferably at least0.1-fold, e.g., at least 0.2-fold, at least 0.3-fold, at least 0.4-fold,at least 0.5-fold, at least 1-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, atleast 50-fold, or at least 100-fold compared to the absence of thedivalent copper cation.

In another aspect, a divalent copper cation increases the thermalstability of a GH61 polypeptide having cellulolytic enhancing activityat least 1.01-fold, e.g., at least 1.05-fold, at least 1.1-fold, atleast 1.5-fold, at least 1.8-fold, at least 2-fold, at least 5-fold, atleast 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, orat least 50-fold compared to the absence of the divalent copper cation.

Polypeptides Having Cellulolytic Enhancing Activity

In the methods of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the GH61 polypeptide having cellulolytic enhancingactivity, comprises the following motifs:

(SEQ ID NO: 65 or SEQ ID NO: 66)[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and[FW]-[TF]-K-[AIV],wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The GH61 polypeptide comprising the above-noted motifs may furthercomprise:

(SEQ ID NO: 67 or SEQ ID NO: 68) H-X(1,2)-G-P-X(3)-[YW]-[AILMV],(SEQ ID NO: 69) [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or(SEQ ID NO: 70 or SEQ ID NO: 71) H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], (SEQ ID NO: 72)wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred embodiment, the GH61 polypeptide having cellulolyticenhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQID NO: 73 or SEQ ID NO: 74). In another preferred embodiment, the GH61polypeptide having cellulolytic enhancing activity further comprises[EQ]X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 75). In anotherpreferred embodiment, the GH61 polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 76or SEQ ID NO: 77) and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ IDNO: 78).

In a second aspect, the GH61 polypeptide having cellulolytic enhancingactivity, comprises the following motif:

(SEQ ID NO: 79 or SEQ ID NO: 80)[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A- [HNQ],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

In a third aspect, the GH61 polypeptide having cellulolytic enhancingactivity comprises or consists of an amino acid sequence having at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, or at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 100% sequence identity to the mature polypeptideof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64.

In a preferred embodiment, the mature polypeptide is amino acids 20 to326 of SEQ ID NO: 2, amino acids 18 to 239 of SEQ ID NO: 4, amino acids20 to 258 of SEQ ID NO: 6, amino acids 19 to 226 of SEQ ID NO: 8, aminoacids 20 to 304 of SEQ ID NO: 10, amino acids 16 to 317 of SEQ ID NO:12, amino acids 22 to 249 of SEQ ID NO: 14, amino acids 20 to 249 of SEQID NO: 16, amino acids 18 to 232 of SEQ ID NO: 18, amino acids 16 to 235of SEQ ID NO: 20, amino acids 19 to 323 of SEQ ID NO: 22, amino acids 16to 310 of SEQ ID NO: 24, amino acids 20 to 246 of SEQ ID NO: 26, aminoacids 22 to 354 of SEQ ID NO: 28, amino acids 22 to 250 of SEQ ID NO:30, amino acids 22 to 322 of SEQ ID NO: 32, amino acids 24 to 444 of SEQID NO: 34, amino acids 26 to 253 of SEQ ID NO: 36, amino acids 18 to 246of SEQ ID NO: 38, amino acids 20 to 334 of SEQ ID NO: 40, amino acids 18to 227 of SEQ ID NO: 42, amino acids 20 to 223 of SEQ ID NO: 44, aminoacids 22 to 368 of SEQ ID NO: 46, amino acids 25 to 330 of SEQ ID NO:48, amino acids 17 to 236 of SEQ ID NO: 50, amino acids 19 to 250 of SEQID NO: 52, amino acids 23 to 478 of SEQ ID NO: 54, amino acids 17 to 230of SEQ ID NO: 56, amino acids 20 to 257 of SEQ ID NO: 58, amino acids 23to 251 of SEQ ID NO: 60, amino acids 19 to 349 of SEQ ID NO: 62, oramino acids 24 to 436 of SEQ ID NO: 64.

In a fourth aspect, the GH61 polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide that hybridizes under at leastvery low stringency conditions, e.g., at least low stringencyconditions, at least medium stringency conditions, at least medium-highstringency conditions, at least high stringency conditions, or at leastvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 15, or the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, or (iii) a full-lengthcomplement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

In a preferred embodiment, the mature polypeptide coding sequence isnucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ IDNO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 ofSEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 46 to951 of SEQ ID NO: 11, nucleotides 64 to 796 of SEQ ID NO: 13,nucleotides 77 to 766 of SEQ ID NO: 15, nucleotides 52 to 921 of SEQ IDNO: 17, nucleotides 46 to 851 of SEQ ID NO: 19, nucleotides 55 to 1239of SEQ ID NO: 21, nucleotides 46 to 1250 of SEQ ID NO: 23, nucleotides58 to 811 of SEQ ID NO: 25, nucleotides 64 to 1112 of SEQ ID NO: 27,nucleotides 64 to 859 of SEQ ID NO: 29, nucleotides 64 to 1018 of SEQ IDNO: 31, nucleotides 70 to 1483 of SEQ ID NO: 33, nucleotides 76 to 832of SEQ ID NO: 35, nucleotides 52 to 875 of SEQ ID NO: 37, nucleotides 58to 1250 of SEQ ID NO: 39, nucleotides 52 to 795 of SEQ ID NO: 41,nucleotides 58 to 974 of SEQ ID NO: 43, nucleotides 64 to 1104 of SEQ IDNO: 45, nucleotides 73 to 990 of SEQ ID NO: 47, nucleotides 49 to 1218of SEQ ID NO: 49, nucleotides 55 to 930 of SEQ ID NO: 51, nucleotides 67to 1581 of SEQ ID NO: 53, nucleotides 49 to 865 of SEQ ID NO: 55,nucleotides 58 to 1065 of SEQ ID NO: 57, nucleotides 67 to 868 of SEQ IDNO: 59, nucleotides 55 to 1099 of SEQ ID NO: 61, or nucleotides 70 to1483 of SEQ ID NO: 63.

In a fifth aspect, the GH61 polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence having at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, or SEQ ID NO: 63; or the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, or SEQ ID NO: 15, or the cDNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQID NO: 63.

In a sixth aspect, the GH61 polypeptide having cellulolytic enhancingactivity is a variant of the mature polypeptide of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ IDNO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,SEQ ID NO: 62, or SEQ ID NO: 64 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide is up to 10, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minornature, that is conservative amino acid substitutions or insertions thatdo not significantly affect the folding and/or activity of the protein;small deletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for cellulolytic enhancingactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem.271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to the parentpolypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

A GH61 polypeptide having cellulolytic enhancing activity may beobtained from microorganisms of any genus. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A GH61 polypeptide having cellulolytic enhancing activity may be abacterial polypeptide. For example, the polypeptide may be a Grampositive bacterial polypeptide such as a Bacillus, Streptococcus,Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,Clostridium, Geobacillus, or Oceanobacillus polypeptide havingcellulolytic enhancing activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide.

In one aspect, the GH61 polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide.

In another aspect, the GH61 polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide.

In another aspect, the GH61 polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide.

The GH61 polypeptide having cellulolytic enhancing activity may be afungal polypeptide. For example, the polypeptide may be a yeastpolypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungalpolypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus,Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide.

In another aspect, the GH61 polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasfi, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide.

In another preferred aspect, the GH61 polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfoetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Penicilliumthomfi, Phanerochaete chrysosporium, Thielavia achromatica, Thielaviaalbomyces, Thielavia albopilosa, Thielavia australeinsis, Thielaviafimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana,Thielavia setosa, Thielavia spededonium, Thielavia subthermophila,Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii,Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, orTrichophaea saccata polypeptide.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) or DNA samples obtained directly from naturalmaterials (e.g., soil, composts, water, etc.) using the above-mentionedprobes. Techniques for isolating microorganisms and DNA directly fromnatural habitats are well known in the art. A polynucleotide encodingthe polypeptide may then be obtained by similarly screening a genomicDNA or cDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

GH61 polypeptides having cellulolytic enhancing activity also includefused polypeptides or cleavable fusion polypeptides in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof having cellulolytic enhancing activity.Techniques for producing fusion polypeptides are known in the art, andinclude ligating the coding sequences encoding the polypeptides so thatthey are in frame and that expression of the fusion polypeptide is undercontrol of the same promoter(s) and terminator. Fusion polypeptides mayalso be constructed using intein technology in which fusion polypeptidesare created post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover(PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid 5-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁶ toabout 1, about 10⁻⁶ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁶ to about 1 g, about 10⁻⁶ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

Cellulolytic Enzyme Compositions

The present invention also relates to enzyme compositions comprising aGH61 polypeptide having cellulolytic enhancing activity and a divalentcopper cation, wherein the divalent copper cation is present at aconcentration of 0.0001 mM to about 20 mM, e.g., about 0.0005 mM toabout 15 mM, about 0.001 mM to about 10 mM, about 0.005 mM to about 5mM, about 0.01 mM to about 2.5 mM, about 0.05 mM to about 1 mM, or about0.1 mM to about 1 mM during degradation or conversion of a cellulosicmaterial and the presence of the divalent copper cation and the GH61polypeptide having cellulolytic enhancing activity increases thedegradation or conversion of the cellulosic material by an enzymecomposition compared to the GH61 polypeptide having cellulolyticenhancing activity without the divalent copper cation.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

The compositions may also be a fermentation broth formulation or a cellcomposition, as described herein. In some embodiments, the compositionis a cell-killed whole broth containing organic acid(s), killed cellsand/or cell debris, and culture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compostions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis. In some embodiments, the cell-killed whole broth orcomposition contains the spent cell culture medium, extracellularenzymes, and killed filamentous fungal cells. In some embodiments, themicrobial cells present in the cell-killed whole broth or compositioncan be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

The enzyme compositions can comprise any protein useful in degrading orconverting the cellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (e.g., several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and a polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and a polypeptide havingcellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., GH61 polypeptides havingcellulolytic enhancing activity, (collectively hereinafter “polypeptideshaving enzyme activity”) can be derived or obtained from any suitableorigin, including, bacterial, fungal, yeast, plant, or mammalian origin.The term “obtained” also means herein that the enzyme may have beenproduced recombinantly in a host organism employing methods describedherein, wherein the recombinantly produced enzyme is either native orforeign to the host organism or has a modified amino acid sequence,e.g., having one or more (e.g., several) amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtainedrecombinantly, such as by site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocaffimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasfi, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™(Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (NovozymesA/S), ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP(Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™7069 W (Rohm GmbH), FIBREZYME® LDI (Dyadic International, Inc.),FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150L (DyadicInternational, Inc.). The cellulase enzymes are added in amountseffective from about 0.001 to about 5.0 wt % of solids, e.g., about0.025 to about 4.0 wt % of solids or about 0.005 to about 2.0 wt % ofsolids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GENBANK™ accession no. M15665),Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22), Trichoderma reesei CeI5A endoglucanase II (GENBANK™ accessionno. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563, GENBANK™ accession no. AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GENBANK™ accession no. Z33381), Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439), Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381), Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_(—)324477), Humicolainsolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6Bendoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase,Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestrisNRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7Fendoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase, and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielaviaterrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichodermareesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,648,263, and U.S. Pat. No. 5,686,593.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458), and Talaromycesemersonii (SwissProt accession number Q8X212).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GWX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q7s259), Phaeosphaerianodorum (Uniprot accession number QOUHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in themethods of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP accession number AAR94170), Humicolainsolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus(WO 2006/114094).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt accession number alccl2), Aspergillusfumigatus (SwissProt accession number Q4WW45), Aspergillus niger(Uniprot accession number Q96WX9), Aspergillus terreus (SwissProtaccession number Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8X211), and Trichoderma reesei (Uniprotaccession number Q99024).

The polypeptides having enzyme activity used in the methods of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Nucleic Acid Constructs

An isolated polynucleotide encoding a polypeptide having enzyme activitymay be manipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the polynucleotide prior to its insertioninto a vector may be desirable or necessary depending on the expressionvector. The techniques for modifying polynucleotides utilizingrecombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide.

The promoter contains transcriptional control sequences that mediate theexpression of the polypeptide. The promoter may be any polynucleotidethat shows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dana (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase Ill,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The various nucleotide and control sequences described herein may bejoined together to produce a recombinant expression vector comprising apolynucleotide encoding a polypeptide having enzyme activity, apromoter, and transcriptional and translational stop signals. Theexpression vectors may include one or more convenient restriction sitesto allow for insertion or substitution of the polynucleotide encodingthe polypeptide at such sites. Alternatively, a polynucleotide encodingsuch a polypeptide may be expressed by inserting the polynucleotidesequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

Recombinant host cells comprising a polynucleotide encoding apolypeptide having enzyme activity operably linked to one or morecontrol sequences that direct the production of the polypeptide can beadvantageously used in the recombinant production of the polypeptide. Aconstruct or vector comprising a polynucleotide is introduced into ahost cell so that the construct or vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier. The term “host cell” encompasses any progeny of a parent cellthat is not identical to the parent cell due to mutations that occurduring replication. The choice of a host cell will to a large extentdepend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

Methods for producing a polypeptide having enzyme activity, comprise (a)cultivating a cell, which in its wild-type form is capable of producingthe polypeptide, under conditions conducive for production of thepolypeptide; and optionally (b) recovering the polypeptide.

Alternatively, methods for producing a polypeptide having enzymeactivity, comprise (a) cultivating a recombinant host cell underconditions conducive for production of the polypeptide; and optionally(b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Methods for Processing Cellulosic Material

The compositions and methods of the present invention can be used tosaccharify a cellulosic material to fermentable sugars and to convertthe fermentable sugars to many useful fermentation products, e.g., fuel,potable ethanol, and/or platform chemicals (e.g., acids, alcohols,ketones, gases, and the like). The production of a desired fermentationproduct from the cellulosic material typically involves pretreatment,enzymatic hydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using processes conventional in the art.Moreover, the methods of the present invention can be implemented usingany conventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on temperature rangeand addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730). During steam pretreatment, hemicellulose acetyl groups arecleaved and the resulting acid autocatalyzes partial hydrolysis of thehemicellulose to monosaccharides and oligosaccharides. Lignin is removedto only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexplosion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, thecellulosic material is mixed with dilute acid, typically H₂SO₄, andwater to form a slurry, heated by steam to the desired temperature, andafter a residence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996, supra;Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999,Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier etal., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperatures in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically using anenzyme composition of the present invention comprising an effectiveamount of a GH61 polypeptide having cellulolytic enhancing activity anddivalent copper cation. The enzyme components of the composition canalso be added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 5.0 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt %, e.g., about 10 toabout 40 wt % or about 20 to about 30 wt %.

The optimum amounts of the enzymes and a GH61 polypeptide(s) havingcellulolytic enhancing activity depend on several factors including, butnot limited to, the mixture of component cellulolytic enzymes and/orhemicellulolytic enzymes, the cellulosic material, the concentration ofcellulosic material, the pretreatment(s) of the cellulosic material,temperature, time, pH, and inclusion of fermenting organism (e.g., yeastfor Simultaneous Saccharification and Fermentation).

In a preferred aspect, an effective amount of cellulolytic orhemicellulolytic enzyme to the cellulosic material is about 0.5 to about50 mg, preferably about 0.5 to about 40 mg, more preferably about 0.5 toabout 25 mg, more preferably about 0.75 to about 20 mg, more preferablyabout 0.75 to about 15 mg, even more preferably about 0.5 to about 10mg, and most preferably about 2.5 to about 10 mg per g of the cellulosicmaterial.

In another preferred aspect, an effective amount of a GH61 polypeptidehaving cellulolytic enhancing activity to cellulosic material is about0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg, morepreferably about 0.01 to about 30 mg, more preferably about 0.01 toabout 20 mg, more preferably about 0.01 to about 10 mg, more preferablyabout 0.01 to about 5 mg, more preferably at about 0.025 to about 1.5mg, more preferably at about 0.05 to about 1.25 mg, more preferably atabout 0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25mg, even more preferably at about 0.15 to about 1.25 mg, and mostpreferably at about 0.25 to about 1.0 mg per g of cellulosic material.

In another preferred aspect, an effective amount of a GH61 polypeptidehaving cellulolytic enhancing activity to cellulolytic enzyme is about0.005 to about 1.0 g, preferably about 0.01 to about 1.0 g, morepreferably about 0.15 to about 0.75 g, more preferably about 0.15 toabout 0.5 g, more preferably about 0.1 to about 0.5 g, even morepreferably about 0.1 to about 0.25 g, and most preferably about 0.05 toabout 0.2 g per g of cellulolytic enzyme.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Preferred yeastincludes strains of Candida, Kluyveromyces, and Saccharomyces, e.g.,Candida sonorensis, Kluyveromyces marxianus, and Saccharomycescerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In another more preferred aspect, the yeast is Saccharomyces cerevisiae.In another more preferred aspect, the yeast is Saccharomyces distaticus.In another more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacillus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coli. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine.

In another more preferred aspect, the amino acid is lysine. In anothermore preferred aspect, the amino acid is serine. In another morepreferred aspect, the amino acid is threonine. See, for example,Richard, A., and Margaritis, A., 2004, Empirical modeling of batchfermentation kinetics for poly(glutamic acid) production and othermicrobial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Preparation of GH61 Polypeptides

Thermoascus aurantiacus GH61A polypeptide and Thielavia terrestris GH61Epolypeptide were prepared according to WO 2005/074656 and WO2005/074647, respectively.

Each GH61 polypeptide sample at approximately 0.5 mM in 10 mM sodiumacetate buffer was thoroughly demetallated using a solid chelating resinaccording to the procedure of Carrer et al., 2006, Anal. Bioanal. Chem.385: 1409-1413, which generates clean apo-GH61 polypeptide with no othermetal species in solution. A 250 μl volume of each of the demetallatedpolypeptides were individually treated with 10 μl of a 10 mM aqueoussolution of copper(II) nitrate. Sufficient metal solution was added tocreate a 1:1 metal:protein stoichiometry with an overall metal-proteinconcentration of approximately 0.5 mM.

In a further experiment the copper-treated T. aurantiacus GH61Apolypeptide was added to a 10 mM ascorbate and 0.1% PASO solution.

Example 2 Electron Paramagnetic Resonance (EPR)

Continuous wave EPR spectra were obtained as frozen glasses in 10-20%glycerol solutions at 140 K using a Bruker EMX spectrometer (Bruker AXSGmbH, Karlsruhe, Germany) at 9.28 GHz.

As a control, an EPR spectrum of 1.0 mM copper(II) nitrate in 10 mMsodium acetate buffer was recorded (FIG. 1), which exhibited a standardanisotropic splitting pattern for a copper(II) species in an axiallyelongated coordination environment. As further controls, the EPR spectraof demetallated T. aurantiacus GH61A polypeptide and T. terrestris GH61Epolypeptide (FIG. 2) showed no discernible signal above backgroundindicating that no paramagnetic species were present in theapo-proteins.

EPR spectra of the copper-treated T. aurantiacus GH61A polypeptide andT. terrestris GH61E polypeptide are shown in FIGS. 3A and 3B,respectively. The spectra were typical of a copper(II) species withclear hyperfine coupling in the parallel dimension to the copper(I=3/2). The two spectra were almost identical but both weresignificantly different from that of a simple aqueous copper(II)solution (FIG. 1). The spectra indicated that a copper-protein complexhad been generated in both T. aurantiacus GH61A polypeptide and T.terrestris GH61E polypeptide and the binding site for each polypeptidewas very similar. Additionally, each spectrum yielded a clearanisotropic signal typical of a single copper(II), i.e., the copperbound to the protein at a single and well-defined site.

Both Cu-T. aurantiacus GH61A polypeptide and Cu-T. terrestris GH61Epolypeptide showed loss of the copper-EPR signal on treatment with 10 mMascorbate and 1% PASO (FIGS. 4A and 4B). A small residual signalattributable to an organic-based radical was seen indicating the copperwas reduced from copper(II) to copper(I).

Example 3 Methods of Evaluating the Effect of Cupric Ion on GH61Polypeptides Having Cellulolytic Enhancing Activity

The effect of cupric (copper(II)) ions on the cellulolytic enhancingactivity of GH61 polypeptides was evaluated according to the proceduresdescribed below.

Microcrystalline cellulose (AVICEL® PH101; Sigma-Aldrich (St. Louis,Mo., USA) was used as the source of cellulosic material.

A Trichoderma reesei cellulase composition (CELLUCLAST® supplementedwith Aspergillus oryzae beta-glucosidase, available from Novozymes A/S,Bagsvaerd, Denmark) was used as the cellulase preparation. The cellulasepreparation is designated herein in the Examples as “Trichoderma reeseicellulase composition”.

The hydrolysis of AVICEL® was conducted using 2.0 ml deep-well plates(Axygen Scientific, Union City, Calif., USA) in a total reaction volumeof 1.0 ml. Each hydrolysis was performed with 14 mg of AVICEL® (14 mg ofcellulose) per ml of 50 mM sodium acetate pH 5.0 buffer, the T. reeseicellulase composition at 4 mg protein per gram of cellulose, with andwithout GH61 polypeptide having cellulolytic enhancing activity at 0.4mg per g cellulose, as well as with and without copper sulfate, anddehydroascorbate at a specified concentration. The plate was then sealedusing an ALPS-300™ or ALPS-3000™ plate heat sealer (Abgene, Epsom,United Kingdom), mixed thoroughly, and incubated at 50° C. for 3-7 daysin an Isotemp Plus incubator (Thermo Fisher Scientific Inc., Waltham,Mass., USA). All experiments were performed at least in duplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples, diluted to appropriate concentrations in0.005 M H₂SO₄, were measured using a 4.6×250 mm AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA) by elution with0.05% (w/w) benzoic acid-0.005 M H₂SO₄ at 65° C. at a flow rate of 0.6ml per minute, and quantitated by integration of the glucose andcellobiose signals from refractive index detection (CHEMSTATION®,AGILENT® 1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA)calibrated by pure sugar samples. The resultant glucose and cellobioseequivalents were used to calculate the percentage of celluloseconversion for each reaction. Measured sugar concentrations wereadjusted for the appropriate dilution factor. Data were processed usingMICROSOFT EXCEL™ software (Microsoft, Richland, Wash., USA).

Percent conversion was calculated based on the mass ratio of solubilizedglucosyl units to the initial mass of insoluble cellulose. Only glucoseand cellobiose were measured for soluble sugars, as cellodextrins longerthan cellobiose were present in negligible concentrations (due toenzymatic hydrolysis). The extent of total cellulose conversion wascalculated using the following equation:

$\begin{matrix}{{\% \mspace{14mu} {conversion}} = {\frac{\left( {{\lbrack{glucose}\rbrack \left( \frac{mg}{ml} \right)} + \left( {1.053 \times \lbrack{cellobiose}\rbrack \left( \frac{mg}{ml} \right)} \right)} \right)}{1.111 \times \lbrack{cellulose}\rbrack \left( \frac{mg}{ml} \right)} \times 100}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

The 1.111 and 1.053 factors for glucose and cellobiose, respectively,take into account the increase in mass when the glucosyl units incellulose (average molecular mass of 162 daltons) are converted toglucose (molecular mass of 180 daltons) or cellobiose glucosyl units(average molecular mass of 171 daltons).

The compounds evaluated were dehydroascorbic acid and copper sulfateobtained from Sigma-Aldrich Co. (St. Louis, Mo., USA).

Example 4 Preparation of T. aurantiacus GH61A Polypeptide HavingCellulolytic Enhancing Activity

T. aurantiacus GH61A polypeptide having cellulolytic enhancing activitywas recombinantly prepared according to WO 2005/074656 using Aspergillusoryzae JaL250 as a host.

The recombinantly produced T. aurantiacus GH61A polypeptide was firstconcentrated from 60 ml to 7 ml, by ultrafiltration using a 10 kDamembrane (VIVASPIN®, GE Healthcare, Piscataway, N.J., USA), bufferexchanged into 20 mM Tris-HCl plus 150 mM NaCl pH 8.0, and then purifiedusing a 320 ml SUPERDEX® 75 column (GE Healthcare, Piscataway, N.J.,USA) equilibrated with 20 mM Tris-HCl plus 150 mM NaCl pH 8.0 at a flowrate of 1 ml per minute. Fractions of 5 ml were collected and pooledbased on SDS-PAGE.

Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit (Thermo Fisher Scientific Inc., Rockford, Ill., USA) in whichbovine serum albumin was used as a protein standard.

Example 5 Effect of T. aurantiacus GH61A Having Cellulolytic EnhancingActivity on Hydrolysis of Microcrystalline Cellulose by the Trichodermareesei Cellulase Composition

The effect of T. aurantiacus GH61A polypeptide (Example 4) on hydrolysisof AVICEL® by the T. reesei cellulase composition (Example 3) wasdetermined using the same experimental conditions and proceduresdescribed in Example 3 in the absence of copper sulfate anddehydroascorbate.

The presence of the T. aurantiacus GH61A polypeptide did not enhance thehydrolysis of AVICEL® by the T. reesei cellulase composition. Percentconversion of AVICEL® was 15.2±0.1%, 29.4±0.4%, and 43.7±0.1% at 1, 3,and 7 days, respectively, in the absence of the T. aurantiacus GH61Apolypeptide compared to 14.5±0.4%, 29.7±0.3%, and 42.6±0.9% at 1, 3, and7 days, respectively, in the presence of the T. aurantiacus GH61Apolypeptide.

Example 6 Effect of Cupric Ion on the Thermoascus aurantiacus GH61APolypeptide During Hydrolysis of Microcrystalline Cellulose by theTrichoderma reesei Cellulase Composition

The effects of copper sulfate on the cellulolytic enhancing activity ofthe T. aurantiacus GH61A polypeptide during hydrolysis of AVICEL® by theT. reesei cellulase composition (Example 3) was determined using theexperimental conditions and procedures described in Example 3 with thefollowing additions. The concentration of copper sulfate was 0, 1, 10,or 100 μM, and the concentration of dehydroascorbate was 0 or 5 mM.

The effect of cupric ion on hydrolysis of a cellulosic material by theT. reesei cellulase composition in the absence of the T. aurantiacusGH61A polypeptide was quantified by determining the ratio of percentconversion of the cellulosic material in the presence of cupric ion tothe percent conversion of the cellulosic material in the absence ofcupric ion:

$\begin{matrix}{{{Cupric}\mspace{14mu} {ion}\mspace{14mu} {effect}_{({{no}\mspace{14mu} {GH}\; 61})}} = \frac{\% \mspace{14mu} {conversion}_{({{{no}\mspace{14mu} {GH}\; 61} + {{cupric}\mspace{14mu} {ion}}})}}{\% \mspace{14mu} {conversion}_{({{no}\mspace{14mu} {GH}\; 61\mspace{14mu} {no}\mspace{14mu} {cupric}\mspace{14mu} {ion}})}}} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$

Stimulation of hydrolysis by cupric ion yields a ratio >1; inhibition ofhydrolysis yields a ratio <1, and no effect on hydrolysis yields aratio=1 (FIGS. 5 and 7, white bars).

The effect of cupric ion on hydrolysis of a cellulosic material by theT. reesei cellulase composition in the presence of the T. aurantiacusGH61A polypeptide was quantified by determining the ratio of percentconversion of the cellulosic material in the presence of cupric ion tothe percent conversion of the cellulosic material in the absence of theheterocyclic compound, i.e., dehydroascorbate:

$\begin{matrix}{{{Cupric}\mspace{14mu} {ion}\mspace{14mu} {effect}_{({{+ {GH}}\; 61})}} = \frac{\% \mspace{14mu} {conversion}_{({{{+ {GH}}\; 61} + {{cupric}\mspace{14mu} {ion}}})}}{\% \mspace{14mu} {conversion}_{({{+ {GH}}\; 61\mspace{14mu} {no}\mspace{14mu} {cupric}\mspace{14mu} {ion}})}}} & \left( {{Equation}\mspace{14mu} 3} \right)\end{matrix}$

Stimulation of hydrolysis by cupric ion in the presence of the GH61polypeptide yields a ratio >1; inhibition of hydrolysis yields a ratio<1, and no effect on hydrolysis yields a ratio=1 (FIGS. 5 and 7, greybars).

The effect of the T. aurantiacus GH61A polypeptide on hydrolysis of acellulosic material by the T. reesei cellulase composition in thepresence of cupric ion was quantified by determining the ratio ofpercent conversion of the cellulosic material in the presence of theGH61 polypeptide to the percent conversion of the cellulosic material inthe absence of the GH61 polypeptide:

$\begin{matrix}{{{GH}\; 61\mspace{14mu} {effect}} = \frac{\% \mspace{14mu} {conversion}_{({{{+ {GH}}\; 61} + {{cupric}\mspace{14mu} {ion}}})}}{\% \mspace{14mu} {conversion}_{({{{no}\mspace{14mu} {GH}\; 61} + {{cupric}\mspace{14mu} {ion}}})}}} & \left( {{Equation}\mspace{14mu} 4} \right)\end{matrix}$

Enhancement of hydrolysis by the GH61 polypeptide yields a ratio >1;inhibition of hydrolysis yields a ratio <1, and no effect on hydrolysisyields a ratio=1 (FIGS. 5 and 7, black bars).

FIG. 5 shows (1) the effect of cupric ion on hydrolysis of AVICEL® bythe T. reesei cellulase composition in the absence of the T. aurantiacusGH61A polypeptide (Cupric ion effect_((no GH61)), white bars), (2) theeffect of cupric ion on hydrolysis of AVICEL® by the T. reesei cellulasecomposition in the presence of the T. aurantiacus GH61A polypeptide(Cupric ion effect_((+GH61)), grey bars), and (3) the effect of the T.aurantiacus GH61A polypeptide on hydrolysis of AVICEL® by the T. reeseicellulase composition in the presence of cupric ion (GH61 effect, blackbars) for 1, 3, and 7 days in the presence of dehydroascorbic acid.

Hydrolysis of AVICEL® by the T. reesei cellulase composition was inertto the presence of low concentration of cupric ion, but inhibited by thepresence of high concentration of cupric ion, as shown by a Cupric ioneffect_((−GH61)) (as defined by Equation 2) less than 1 (FIG. 5, whitebars). The presence of the T. aurantiacus GH61A polypeptide alleviatedthe cupric ion inhibition (FIG. 5, grey bars), as shown by a Cupric ioneffect_((+GH61)) (as defined by Equation 3) close to 1. Furthermore, theeffect of the T. aurantiacus GH61A polypeptide was greater than 1 (GH61effect, Equation 4), indicating that the T. aurantiacus GH61Apolypeptide enhanced hydrolysis when cupric ion was present (FIG. 5,black bars). FIG. 6 shows that the cupric ion's activation of the T.aurantiacus GH61A polypeptide's enhancement depends on cupric ionconcentration. The T. aurantiacus GH61A polypeptide did not enhancehydrolysis of microcrystalline cellulose in the absence of cupric ionand dehydroascorbic acid (Example 3).

FIG. 7 shows (1) the effect of cupric ion on hydrolysis of AVICEL® bythe T. reesei cellulase composition in the absence of the T. aurantiacusGH61A polypeptide (Cupric ion effect_((no GH61)), white bars), (2) theeffect of cupric ion on hydrolysis of AVICEL® by the T. reesei cellulasecomposition in the presence of the T. aurantiacus GH61A polypeptide(Cupric ion effect_((+GH61)), grey bars), and (3) the effect of the T.aurantiacus GH61A polypeptide on hydrolysis of AVICEL® by the T. reeseicellulase composition in the presence of cupric ion (GH61 effect, blackbars) for 1, 3, and 7 days, in the absence of dehydroascorbic acid.

Hydrolysis of AVICEL® by the T. reesei cellulase composition wasslightly enhanced by the presence of low concentration of cupric ion,but inhibited by the presence of high concentration of cupric ion, asshown by a Cupric ion effect_((−GH61)) (as defined by Equation 2) lessthan 1 (FIG. 7, white bars). The presence of the T. aurantiacus GH61Apolypeptide partially alleviated the cupric ion inhibition (FIG. 7, greybars), as shown by a Cupric ion effect_((+GH61)) (as defined by Equation3) closer to 1 than the Cupric ion effect_((−GH61)). Furthermore, theeffect of the T. aurantiacus GH61A polypeptide was greater than 1 (GH61effect, Equation 4), indicating that the T. aurantiacus GH61Apolypeptide enhanced hydrolysis when cupric ion was present (FIG. 7,black bars). FIG. 8 shows that the cupric ion's activation of the T.aurantiacus GH61A polypeptide's enhancement depended on cupric ionconcentration. T. aurantiacus GH61A polypeptide did not enhancehydrolysis of microcrystalline cellulose in the absence of cupric ionand dehydroascorbic acid (Example 3).

The overall results demonstrated that cellulolytic enhancing activity ofthe T. aurantiacus GH61A polypeptide was apparent in the presence ofcupric ion during hydrolysis of AVICEL® by the T. reesei cellulasecomposition. However, the T. aurantiacus GH61A polypeptide had nodetectable effect on hydrolysis of AVICEL® by the T. reesei cellulasecomposition in the absence of cupric ion.

Example 7 Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose, and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Milled unwashed PCS (dry weight 32.35%) was prepared by milling wholeslurry PCS in a Cosmos ICMG 40 wet multi-utility grinder (EssEmmCorporation, Tamil Nadu, India).

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of insoluble PCS solids per mlof 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfateand the indicated protein loading of the enzyme composition (expressedas mg protein per gram of cellulose). Enzyme compositions were preparedand then added simultaneously to all wells in a volume ranging from 50μl to 200 μl, for a final volume of 1 ml in each reaction. The plateswere then sealed using an ALPS-300™ plate heat sealer (Abgene, Epsom,United Kingdom), mixed thoroughly, and incubated at a specifictemperature for 72 hours. All experiments reported were performed intriplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65°C. at a flow rate of 0.6 ml per minute, and quantitation by integrationof the glucose, cellobiose, and xylose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantglucose and cellobiose equivalents were used to calculate the percentageof cellulose conversion for each reaction.

Glucose, cellobiose, and xylose were measured individually. Measuredsugar concentrations were adjusted for the appropriate dilution factor.In case of unwashed PCS, the net concentrations ofenzymatically-produced sugars were determined by adjusting the measuredsugar concentrations for corresponding background sugar concentrationsin unwashed PCS at zero time points. All HPLC data processing wasperformed using MICROSOFT EXCEL™ software (Microsoft, Richland, Wash.,USA).

The degree of cellulose conversion to glucose was calculated using thefollowing equation: % conversion=(glucose concentration/glucoseconcentration in a limit digest)×100. In order to calculate %conversion, a 100% conversion point was set based on a cellulase control(100 mg of Trichoderma reesei cellulase per gram cellulose), and allvalues were divided by this number and then multiplied by 100.Triplicate data points were averaged and standard deviation wascalculated.

Example 8 Preparation of High-Temperature Enzyme Composition

Preparation of Aspergillus fumigatus strain NN055679 GH7Acellobiohydrolase I. The Aspergillus fumigatus GH7A cellobiohydrolase I(SEQ ID NO: 81 [DNA sequence] and SEQ ID NO: 82 [deduced amino acidsequence]) was prepared recombinantly in Aspergillus oryzae as describedin WO 2011/057140. The Aspergillus fumigatus GH7A cellobiohydrolase Iwas purified according to WO 2011/057140.

Preparation of Aspergillus fumigatus strain NN055679 GH6Acellobiohydrolase II. The Aspergillus fumigatus GH6A cellobiohydrolaseII (SEQ ID NO: 83 [DNA sequence] and SEQ ID NO: 84 [deduced amino acidsequence]) was prepared recombinantly in Aspergillus oryzae as describedin WO 2011/057140. The filtered broth of Aspergillus fumigatus GH6Acellobiohydrolase II was buffer exchanged into 20 mM Tris pH 8.0 using a400 ml SEPHADEX™ G-25 column (GE Healthcare, United Kingdom) accordingto the manufacturer's instructions. The fractions were pooled andadjusted to 1.2 M ammonium sulphate-20 mM Tris pH 8.0. The equilibratedprotein was loaded onto a PHENYL SEPHAROSE™ 6 Fast Flow column (highsub) (GE Healthcare, Piscataway, N.J., USA) equilibrated in 20 mM TrispH 8.0 with 1.2 M ammonium sulphate, and bound proteins were eluted with20 mM Tris pH 8.0 with no ammonium sulphate. The fractions were pooled.

Preparation of Thermoascus aurantiacus strain CGMCC 0670 GH5Aendoglucanase II. The Thermoascus aurantiacus GH5A endoglucanase II (SEQID NO: 85 [DNA sequence] and SEQ ID NO: 86 [deduced amino acidsequence]) was prepared recombinantly in Aspergillus oryzae as describedin WO 2011/057140. The Thermoascus aurantiacus GH5A endoglucanase II waspurified according to WO 2011/057140.

Preparation of Aspergillus fumigatus strain NN055679 GH10 xylanase. TheAspergillus fumigatus GH10 xylanase (xyn3) (SEQ ID NO: 87 [DNA sequence]and SEQ ID NO: 88 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2(WO 2000/39322) as a host. The filtered broth of Aspergillus fumigatusNN055679 GH10 xylanase (xyn3) was desalted and buffer-exchanged into 50mM sodium acetate pH 5.0 using a HIPREP® 26/10 Desalting Columnaccording to the manufacturer's instructions.

Preparation of Aspergillus fumigatus strain NN055679 GH3Abeta-glucosidase. The Aspergillus fumigatus GH3A beta-glucosidase (SEQID NO: 89 [DNA sequence] and SEQ ID NO: 90 [deduced amino acidsequence]) was recombinantly prepared according to WO 2005/047499 usingAspergillus oryzae as a host. The filtered broth was adjusted to pH 8.0with 20% sodium acetate, which made the solution turbid. To remove theturbidity, the solution was centrifuged (20000×g, 20 minutes), and thesupernatant was filtered though a 0.2 μm filtration unit (Nalgene,Rochester, N.Y., USA). The filtrate was diluted with deionized water toreach the same conductivity as 50 mM Tris/HCl, pH 8.0. The adjustedenzyme solution was applied to a Q SEPHAROSE® Fast Flow column (GEHealthcare, Piscataway, N.J., USA) equilibrated in 50 mM Tris-HCl, pH8.0 and eluted with a linear gradient from 0 to 500 mM sodium chloride.Fractions were pooled and treated with 1% (w/v) activated charcoal toremove color from the beta-glucosidase pool. The charcoal was removed byfiltration of the suspension through a 0.2 μm filtration unit (Nalgene,Rochester, N.Y., USA). The filtrate was adjusted to pH 5.0 with 20%acetic acid and diluted 10 times with deionized water. The adjustedfiltrate was applied to SP SEPHAROSE® Fast Flow column (GE Healthcare,Piscataway, N.J., USA) equilibrated in 10 mM succinic acid, pH 5.0 andeluted with a linear gradient from 0 to 500 mM sodium chloride.

Preparation of Talaromyces emersonii CBS 393.64 beta-xylosidase. TheTalaromyces emersonii beta-xylosidase (SEQ ID NO: 91 [DNA sequence] andSEQ ID NO: 92 [deduced amino acid sequence]) was prepared recombinantlyaccording to Rasmussen et al., 2006, Biotechnology and Bioengineering94: 869-876 using Aspergillus oryzae JaL355 as a host (WO 2003/070956).The Talaromyces emersonii beta-xylosidase was purified according toRasmussen et al., 2006, supra.

The protein concentration for each of the monocomponents described abovewas determined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard. A high-temperature enzymecomposition was composed of each monocomponent, prepared as describedabove, as follows: 43.5% Aspergillus fumigatus GH7A cellobiohydrolase I,29.4% Aspergillus fumigatus GH6A cellobiohydrolase II, 11.8% Thermoascusaurantiacus GH5A endoglucanase II, 5.9% Aspergillus fumigatus GH10xylanase (xyn3), 5.9% Aspergillus fumigatus GH3A beta-glucosidase, and3.5% Talaromyces emersonii beta-xylosidase. The high-temperature enzymecomposition is designated herein as “high-temperature enzymecomposition”.

Example 9 Thermal Stability of Thermoascus aurantiacus GH61A Polypeptidein the Presence of Calcium Chloride or Copper Sulfate

The thermal stability of the Thermoascus aurantiacus GH61A polypeptidehaving cellulolytic enhancing activity was compared at 1 g/l at 4° C.,50° C., 55° C., 60° C., and 65° C. for 72 hours in 50 mM sodium acetatepH 5.0 buffer in the presence of 1 mM calcium chloride or 50 μM coppersulfate. For each condition above, residual activity was determined bythe PCS assay according to Example 7 by adding 0.5 mg protein per gcellulose of the Thermoascus aurantiacus GH61A polypeptide to 3.0 mgprotein per g cellulose of the high-temperature enzyme composition(Example 8) at 50° C. The 1 ml reactions with 5% milled unwashed PCSwere conducted for 72 hours in 50 mM sodium acetate pH 5.0 buffercontaining 1 mM manganese sulfate. All reactions were performed intriplicate and involved single mixing at the beginning of hydrolysis.

The results as shown in FIGS. 9A and 9B demonstrated that the T.aurantiacus GH61A polypeptide incubated with copper sulfate retainedalmost all residual activity when incubated at all temperatures whilethe T. aurantiacus GH61A polypeptide incubated with calcium chloride hada significant loss of residual activity at 60° C. and 65° C. with only20% residual activity when incubated at 65° C. The results showed thatthe addition of copper sulfate significantly increased the thermalstability of the Thermoascus aurantiacus GH61A polypeptide compared tothe addition of calcium chloride.

Example 10 Preparation of Thielavia terrestris GH61E, Aspergillusfumigatus GH61B, Penicillium sp. (Emersonii) GH61A, Thermoascuscrustaceus GH61A, Thermoascus aurantiacus GH61A Polypeptides

Thielavia terrestris GH61E polypeptide (SEQ ID NO: 7 [DNA sequence] andSEQ ID NO: 8 [deduced amino acid sequence]) was prepared according to WO2005/074647. To purify the T. terrestris GH61E polypeptide, afermentation culture medium adjusted to 1.5 M ammonium sulfate wasloaded onto a Fast Protein Liquid Chromatography device (FPLC) and aPhenyl SEPHAROSE® HP column (GE Healthcare, Piscataway, N.J., USA)preequilibrated with 20 mM Tris-HCl and 1 M ammonium sulfate pH 9,eluted with 20 mM Tris-HCl pH 9, and fractions of 10 ml were collected.Selected fractions were loaded onto a Sephadex G-25 superfine column (GEHealthcare, Piscataway, N.J., USA) preequilibrated with 20 mM Tris-HClpH 9 and eluted with the same buffer for de-salting/buffer-exchange. Thecollected samples were loaded onto a Source 30Q column (GE Healthcare,Piscataway, N.J., USA) preequilibrated with 20 mM Tris-HCl pH 9 andeluted with 20 mM Tris-HCl pH 9 and 1 M NaCl, and the purified GH61polypeptide was collected in 5 ml fractions. Aspergillus fumigatus GH61Bpolypeptide (SEQ ID NO: 29 [DNA sequence] and

SEQ ID NO: 30 [deduced amino acid sequence]) was prepared according toWO 2010/138754. To purify A. fumigatus GH61B polypeptide, a desalted andconcentrated fermentation culture medium was loaded onto a FPLC and aMONO Q® column (GE Healthcare, Piscataway, N.J., USA) preequilibratedwith 20 mM Tris-HCl pH 8, eluted with 20 mM Tris-HCl pH 8 and 1 M NaCl,and fractions of 6 ml were collected. Selected fractions were pooled andconcentrated using molecular weight cut-off 5 kD VIVASPIN® filters (GEHealthcare, Piscataway, N.J., USA), then loaded onto a Superdex 75 HighLoad 26/60 column (GE Healthcare, Piscataway, N.J., USA) preequilibratedwith 20 mM MES-NaOH pH 6, and eluted with the same buffer. The purifiedGH61 polypeptide was collected in 6 ml fractions. The purified fractionswere concentrated using a VIVASPIN® filter.

Penicillium sp. (emersonii) GH61A polypeptide (SEQ ID NO: 35 [DNAsequence] and SEQ ID NO: 36 [deduced amino acid sequence]) was preparedaccording to WO 2011/041397. To purify P. emersonii GH61A polypeptide, afermentation culture medium was loaded onto a FPLC and a Sephadex G-25column (GE Healthcare, Piscataway, N.J., USA) preequilibrated with 20 mMTris.HCl pH 8.0, and eluted with the same buffer to de-salt. Thebuffer-exchanged sample was loaded onto a Q SEPHAROSE® Big Beads column(GE Healthcare, Piscataway, N.J., USA) preequilibrated with 20 mMTris.HCl, pH 7.5, eluted with 20 mM Tris-HCl pH 7.5 and 1 M NaCl, andcollected in 10 ml fractions. Selected fractions were pooled, made in1.5 M ammonium sulfate, and loaded onto a Phenyl SEPHAROSE® HP columnpreequilibrated with 20 mM Tris.HCl, pH 7.5 and 1.5 M ammonium sulfate,eluted with 20 mM Tris.HCl, pH 7.5, and fractions of 12 ml werecollected. Selected fractions were pooled and concentrated using aVIVASPIN® filter, loaded onto a Superdex 75 26/60 column preequilibratedwith 20 mM MES pH 6.0 and 125 mM NaCl, eluted with the same buffer, andfractions of 3 ml were collected. Purified GH61 polypeptide fractionswere pooled and concentrated using a VIVASPIN® filter.

Thermoascus crustaceus GH61A polypeptide (SEQ ID NO: 59 [DNA sequence]and SEQ ID NO: 60 [deduced amino acid sequence]) was prepared accordingto WO 2011/041504. To purify the T. crustaceus GH61A polypeptide, afermentation culture medium was buffer-exchanged and concentrated usingan Ultrareservoir 5L with an Omega 10 kDA MWCO membrane (Pall Filtron,Port Washington, N.Y., USA). The treated sample was loaded onto a FPLCand Q SEPHAROSE® Big Beads column preequilibrated with 20 mM Tris-HCl,pH 8, eluted with 20 mM Tris-HCl pH 8 and 1 M NaCl, and fractions of 12ml were collected. Selected fractions were pooled, adjusted to 1.5 Mammonium sulfate, and loaded onto a Phenyl SEPHAROSE® HP columnpreequilibrated with 20 mM Tris.HCl pH 7.5 and 1.5 M ammonium sulfate.The protein was eluted with 20 mM Tris-HCl, pH 7.5 and reactions of 12ml were collected. Selected fractions were pooled and concentrated usinga Vivacell 100 filter (Vivaproducts, Littleton, Mass., USA). Theconcentrated pooled fractions were loaded onto a Superdex 75 26/60column preequilibrated with 20 mM MES pH 6.0 with 125 mM NaCl, elutedwith the same buffer, and fractions of 3 ml were collected. PurifiedGH61 polypeptide fractions were pooled and concentrated using aVIVASPIN® filter and buffer-exchanged to 20 mM MES pH 6.0.

Thermoascus aurantiacus GH61A polypeptide (SEQ ID NO: 13 [DNA sequence]and SEQ ID NO: 14 [deduced amino acid sequence]) was prepared accordingto WO 2005/074656. To purify the T. aurantiacus GH61A, a fermentationculture medium was buffer-exchanged to 20 mM Tris-HCl pH 8 andconcentrated using a Vivacell 100 filter (Vivaproducts, Littleton,Mass., USA). The treated sample was loaded onto a FPLC and Q SEPHAROSE®Fast Flow column preequilibrated with 20 mM Tris-HCl, pH 8, eluted with20 mM Tris-HCl pH 8 and 1 M NaCl, and collected in 10 ml fractions.Selected fractions were pooled, made in 1.5 M ammonium sulfate, andloaded onto a Phenyl SEPHAROSE® HP column preequilibrated with 20 mMTris.HCl, pH 7.5 and 1.5 M ammonium sulfate, eluted with a gradient of20 mM Tris.HCl pH 7.5, and fractions of 10 ml were collected. Selectedfractions were washed and concentrated with a Pall Minimate TFF Systemwith an Omega 10K membrane (Port Washington, N.Y., USA), loaded onto aSuperdex 75 26/60 column preequilibrated with 20 mM MES pH 6.0 and 125mM NaCl, eluted with the same buffer, and fractions of 3 ml werecollected. Purified GH61 polypeptide fractions were pooled andconcentrated using a Pall Minimate TFF System with an Omega 10Kmembrane.

Example 11 Methods of Evaluating the Effect of Cupric Ion on GH61Polypeptides Thermal Stability

Differential scanning calorimetry (DSC) measurements were carried outusing a capillary DSC instrument (MicroCal, Northampton, Mass., USA).Feedback mode was passive and a cell wash was carried out after eachscan.

Representative assay solutions comprised 50 μl of 0.1 M sodium acetatepH 5, 0.1 M Tris-HCl pH 7, or 0.1 M Tris-HCl pH 9, 25 μl of GH61polypeptide stock solution, and 425 μl of Milli-Q water (Millipore,Billerica, Mass., USA). For copper(II) addition, 20-75 μl of 1 mM coppersulfate were used to replace the same volumes of deionized water. Fordiethylene triamine pentaacetic acid (DTPA) chelator addition, 25 μl of1 mM DTPA were used to replace the same volume of deionized water.

To a final concentration of 0.3-1.5 mg/ml, GH61 polypeptide was addedlast to the reaction mixture, 400 μl of which, along with referencescontaining no GH61 protein, were transferred to a 96 well plate. Theplate was then placed in a thermostated autosampler compartment at 10°C. for a maximum of 48 hours. To ensure that the storage time did notaffect the results, test scans of an identical sample were performedafter 1 hour and after 48 hours.

A scan rate of 1.5° C. per minute was used unless otherwise indicated.The thermal unfolding temperature T_(m) was determined as thetemperature at the peak maximum of the transition from the folded tounfolded state in the obtained thermogram. The Origin software package(MicroCal, GE Healthcare, UK) was used for baseline subtraction andgraph presentation.

Example 12 Effect of Cupric Ion Addition on the Thermal Stability of theThielavia Terrestris GH61E Polypeptide

The thermal stability of the T. terrestris GH61E polypeptide, with orwithout the addition of exogenous copper (II) ion, was measured by thefolding-unfolding transition determined by DSC using the sameexperimental conditions and procedures described in Example 11.

A clear thermal unfolding temperature T_(m) was observed for the T.terrestris GH61E polypeptide. When 1 mM DTPA chelator was added to 1.3g/l of the T. terrestris GH61E polypeptide, no change in T_(m) was seen.When 2 mM Ca(II) was added (at 2:1 molar ratio to DTPA), no change inT_(m) was seen. When 2 mM Cu(II) was added (at 2:1 molar ratio to DTPA),an about 16° C. increase in T_(m) was observed, indicating enhancedthermal stability (FIG. 10).

In another experiment, the presence of 100 μM Cu(II) increased the T_(m)about 18, 18, and 13° C. at pH 5, 7, and 9, respectively, indicatingenhanced thermal stability (FIG. 11).

In another experiment, Cu(II) was added incrementally (by 5 μM) from 40to 150 μM, to a solution of about 65 μM T. terrestris GH61E polypeptide.A Cu concentration dependent up-shift of T_(m) was observed, indicatingenhanced thermal stability and an approximate 1:1 Cu binding by theprotein.

Example 13 Effect of Cupric Ion Addition on the Thermal Stability of theAspergillus Fumigatus GH61B Polypeptide

The thermal stability of the A. fumigatus GH61B polypeptide, with orwithout the addition of exogenous copper (II) ion, was measured by thefolding-unfolding transition determined by DSC using the sameexperimental conditions and procedures described in Example 11.

A clear thermal thermal unfolding temperature T_(m) was observed for theA. fumigatus GH61B polypeptide. When 100 μM Cu(II) was added to amixture of 0.5 g/l A. fumigatus GH61B polypeptide and 1 mM DTPAchelator, an about 13, 12, and 10° C. increase in T_(m) at pH 5, 7, and9, respectively, was observed, indicating enhanced thermal stability(FIG. 12).

Example 14 Effect of Cupric Ion Addition on the Thermal Stability of thePenicillium sp. (Emersonii) GH61A Polypeptide

The thermal stability of the P. emersonii GH61A polypeptide, with orwithout the addition of exogenous copper (II) ion, was measured by thefolding-unfolding transition determined by DSC using the sameexperimental conditions and procedures described in Example 11.

A clear thermal thermal unfolding temperature T_(m) was observed for theP. emersonii GH61A polypeptide. When 100 μM Cu(II) was added to amixture of 0.8 g/l of the P. emersonii GH61A polypeptide and 1 mM DTPAchelator, an about 8, 11, and 9° C. increase in T_(m) at pH 5, 7, and 9,respectively, was observed, indicating enhanced thermal stability (FIG.13).

Example 15 Effect of Cupric Ion Addition on the Thermal Stability of theThermoascus Crustaceus GH61A Polypeptide

The thermal stability of the T. crustaceus GH61A polypeptide, with orwithout the addition of exogenous copper (II) ion, was measured by thefolding-unfolding transition determined by DSC using the sameexperimental conditions and procedures described in Example 11.

A clear thermal thermal unfolding temperature T_(m) was observed for theT. crustaceus GH61A polypeptide. When 100 μM Cu(II) was added to amixture of 0.5 g/l of the T. crustaceus GH61A polypeptide and 1 mM DTPAchelator, an about 11, 11, and 9° C. increase in T_(m) at pH 5, 7, and9, respectively, was observed, indicating enhanced thermal stability(FIG. 14).

Example 16 Effect of Cupric Ion Addition on the Thermal Stability of theThermoascus Aurantiacus GH61A Polypeptide

The thermal stability of the T. aurantiacus GH61A polypeptide, with orwithout the addition of exogenous copper (II) ion, was measured by thefolding-unfolding transition determined by DSC using the sameexperimental conditions and procedures described in Example 11.

A clear thermal thermal unfolding temperature T_(m) was observed for theT. aurantiacus GH61A polypeptide. When 100 μM Cu(II) was added to amixture of 0.8 g/l T. aurantiacus GH61A polypeptide and 1 mM DTPAchelator, an about 7, 9, and 7° C. increase in T_(m) at pH 5, 7, and 9,respectively, was observed, indicating enhanced thermal stability (FIG.15).

Example 17 Effect of CuSO₄ on the Transformation of Methylene Blue inthe Presence of the Thermoascus aurantiacus GH61A Polypeptide andPyrogallol

The activity assay was performed in 96-wells plate using a microplatereader from Spectra Max M2 (Molecular Devices, Sunnyvale, Calif., USA).Temperature of the microplate reader was set at 37° C. The reactionmixture consisted of 20 μl of 500 mM MOPS/NaOH pH 7.0 buffer, 20 μl of 1mM methylene blue (dissolved in MilliQ water), 20 μl of 40 mM pyrogallol(in MilliQ water), appropriate volume (1.6, 4, 8, 16, 40, 80 μl) of a2500 μM stock of CuSO₄ to the respective concentrations (0 to 1000 μM),25 μl of 810 μg/ml of T. aurantiacus GH61A polypeptide, and finallyMilliQ water was added to a final volume of 200 μl. The reaction wasinitiated by addition of pyrogallol and monitored at 400 nm. Forcontrols, all the components were included except the GH61 polypeptide.After a 15 minute reaction, absorbance readings at 400 nm were made andcorrected by deducting the absorbance of the controls.

The results as shown in FIG. 16 demonstrated that the presence of the T.aurantiacus GH61A polypeptide and pyrogallol transformed methylene blueoptimally at approximately 200 μM CuSO₄.

The present invention is further described by the following numberedparagraphs:

[1] A method of increasing the activity of a GH61 polypeptide havingcellulolytic enhancing activity, comprising: adding a divalent coppercation to a composition comprising the GH61 polypeptide, wherein thedivalent copper cation is present at a concentration of 0.0001 mM toabout 20 mM, e.g., about 0.0005 mM to about 15 mM, about 0.001 mM toabout 10 mM, about 0.005 mM to about 5 mM, about 0.01 mM to about 2.5mM, about 0.05 mM to about 1 mM, or about 0.1 mM to about 1 mM duringdegradation or conversion of a cellulosic material.

[2] A method of increasing the stability of a GH61 polypeptide havingcellulolytic enhancing activity, comprising: adding a divalent coppercation to a composition comprising the GH61 polypeptide, wherein thedivalent copper cation is present at a concentration of 0.0001 mM toabout 20 mM, e.g., about 0.0005 mM to about 15 mM, about 0.001 mM toabout 10 mM, about 0.005 mM to about 5 mM, about 0.01 mM to about 2.5mM, about 0.05 mM to about 1 mM, or about 0.1 mM to about 1 mM.

[3] A method of increasing the activity and the stability of a GH61polypeptide having cellulolytic enhancing activity, comprising: adding adivalent copper cation to a composition comprising the GH61 polypeptide,wherein the divalent copper cation is present at a concentration of0.0001 mM to about 20 mM, e.g., about 0.0005 mM to about 15 mM, about0.001 mM to about 10 mM, about 0.005 mM to about 5 mM, about 0.01 mM toabout 2.5 mM, about 0.05 mM to about 1 mM, or about 0.1 mM to about 1mM.

[4] The method of any of paragraphs 1-3, wherein the GH61 polypeptidehaving cellulolytic enhancing activity is selected from the groupconsisting of: (a) a polypeptide having cellulolytic enhancing activitycomprising [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ IDNO: 65 or SEQ ID NO: 66) and [FW]-[TF]-K-[AIV], wherein X is any aminoacid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(4)is any amino acid at 4 contiguous positions; and (b) a polypeptidehaving cellulolytic enhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A-[HNQ] (SEQ ID NO: 79 orSEQ ID NO: 80), wherein X is any amino acid, X(4,5) is any amino acid at4 or 5 contiguous positions, and X(3) is any amino acid at 3 contiguouspositions; wherein the polypeptide having cellulolytic enhancingactivity comprising [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ](SEQ ID NO: 65 or SEQ ID NO: 66) and [FW]-[TF]-K-[AIV] optionallyfurther comprises: H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 67 or SEQID NO: 68), [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 69), orH-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 70 or SEQ ID NO: 71) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 72), wherein X isany amino acid, X(1,2) is any amino acid at 1 position or 2 contiguouspositions, X(3) is any amino acid at 3 contiguous positions, and X(2) isany amino acid at 2 contiguous positions.

[5] The method of any of paragraphs 1-3, wherein the GH61 polypeptidehaving cellulolytic enhancing activity is selected from the groupconsisting of: (a) a polypeptide comprising or consisting of an aminoacid sequence having at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, or at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ IDNO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO:64; (b) a polypeptide encoded by a polynucleotide that hybridizes underat least low stringency conditions, e.g., at least medium stringencyconditions, at least medium-high stringency conditions, at least highstringency conditions, or at least very high stringency conditions with(i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO:51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ IDNO: 61, or SEQ ID NO: 63, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, or SEQ ID NO: 15, or the cDNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQID NO: 63, or (iii) a full-length complement of (i) or (ii); (c) apolypeptide encoded by a polynucleotide comprising or consisting of anucleotide sequence having at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, or SEQ ID NO: 63, or the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, or SEQ ID NO: 15, or the cDNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQID NO: 63; (d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ IDNO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,SEQ ID NO: 62, or SEQ ID NO: 64 comprising a substitution, deletion,and/or insertion at one or more positions; and (e) a polypeptide havingcellulolytic enhancing activity comprising or consisting of the maturepolypeptide of of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ IDNO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64;or a fragment thereof having cellulolytic enhancing activity.

[6] The method of any of paragraphs 1-5, wherein the composition furthercomprises one or more enzymes selected from the group consisting of acellulase, a hemicellulase, an expansin, an esterase, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[7] The method of paragraph 6, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[8] The method of paragraph 6, wherein the hemicellulase is one or moreenzymes selected from the group consisting of a xylanase, an acetyxylanesterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, anda glucuronidase.

[9] The method of any of paragraphs of 1-8, further comprisingsupplementing the concentration of the divalent copper cation tomaintain the effective concentration of the divalent copper cation atabout 0.0001 mM to about 20 mM, e.g., about 0.0005 mM to about 15 mM,about 0.001 mM to about 10 mM, about 0.005 mM to about 5 mM, about 0.01mM to about 2.5 mM, about 0.05 mM to about 1 mM, or about 0.1 mM toabout 1 mM.

[10] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositioncomprising a GH61 polypeptide having cellulolytic enhancing activity anda divalent copper cation, wherein the divalent copper cation is presentat a concentration of about 0.0001 mM to about 20 mM, e.g., about 0.0005mM to about 15 mM, about 0.001 mM to about 10 mM, about 0.005 mM toabout 5 mM, about 0.01 mM to about 2.5 mM, about 0.05 mM to about 1 mM,or about 0.1 mM to about 1 mM.

[11] The method of paragraph 10, wherein the GH61 polypeptide havingcellulolytic enhancing activity is selected from the group consistingof: (a) a polypeptide having cellulolytic enhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ ID NO: 65 or SEQID NO: 66) and [FW]-[TF]-K-[AIV], wherein X is any amino acid, X(4,5) isany amino acid at 4 or 5 contiguous positions, and X(4) is any aminoacid at 4 contiguous positions; and (b) a polypeptide havingcellulolytic enhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A-[HNQ] (SEQ ID NO: 79 orSEQ ID NO: 80), wherein X is any amino acid, X(4,5) is any amino acid at4 or 5 contiguous positions, and X(3) is any amino acid at 3 contiguouspositions; wherein the polypeptide having cellulolytic enhancingactivity comprising [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ](SEQ ID NO: 65 or SEQ ID NO: 66) and [FW]-[TF]-K-[AIV] optionallyfurther comprises: H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 67 or SEQID NO: 68), [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 69), orH-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 70 or SEQ ID NO: 71) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 72), wherein X isany amino acid, X(1,2) is any amino acid at 1 position or 2 contiguouspositions, X(3) is any amino acid at 3 contiguous positions, and X(2) isany amino acid at 2 contiguous positions.

[12] The method of paragraph 10, wherein the GH61 polypeptide havingcellulolytic enhancing activity is selected from the group consistingof: (a) a polypeptide comprising or consisting of an amino acid sequencehaving at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, or at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46,SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leastlow stringency conditions, e.g., at least medium stringency conditions,at least medium-high stringency conditions, at least high stringencyconditions, or at least very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ IDNO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, orSEQ ID NO: 63, (ii) the genomic DNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ IDNO: 15, or the cDNA sequence of the mature polypeptide coding sequenceof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, or(iii) a full-length complement of (i) or (ii); (c) a polypeptide encodedby a polynucleotide comprising or consisting of a nucleotide sequencehaving at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ IDNO: 63, or the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 15,or the cDNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ IDNO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO:47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63; (d) a variant ofthe mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ IDNO: 64 comprising a substitution, deletion, and/or insertion at one ormore positions; and (e) a polypeptide having cellulolytic enhancingactivity comprising or consisting of the mature polypeptide of of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ IDNO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64; or a fragment thereof havingcellulolytic enhancing activity.

[13] The method of any of paragraphs 10-12, wherein the cellulosicmaterial is pretreated.

[14] The method of paragraphs 10-13, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a hemicellulase, an expansin, an esterase, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[15] The method of paragraph 14, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[16] The method of paragraph 14, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[17] The method of any of paragraphs 10-16, further comprisingrecovering the degraded cellulosic material.

[18] The method of paragraph 17, wherein the degraded cellulosicmaterial is a sugar.

[19] The method of paragraph 18, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[20] The method of any of paragraphs 10-19, further comprisingsupplementing the concentration of the divalent copper cation tomaintain the concentration of the divalent copper cation at about 0.0001mM to about 20 mM, e.g., about 0.0005 mM to about 15 mM, about 0.001 mMto about 10 mM, about 0.005 mM to about 5 mM, about 0.01 mM to about 2.5mM, about 0.05 mM to about 1 mM, or about 0.1 mM to about 1 mM.

[21] A method for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme compositioncomprising a GH61 polypeptide having cellulolytic enhancing activity anda divalent copper cation, wherein the divalent copper cation is presentat a concentration of about 0.0001 mM to about 20 mM, e.g., about 0.0005mM to about 15 mM, about 0.001 mM to about 10 mM, about 0.005 mM toabout 5 mM, about 0.01 mM to about 2.5 mM, about 0.05 mM to about 1 mM,or about 0.1 mM to about 1 mM; (b) fermenting the saccharifiedcellulosic material with one or more (e.g., several) fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.

[22] The method of paragraph 21, wherein the GH61 polypeptide havingcellulolytic enhancing activity is selected from the group consistingof: (a) a polypeptide having cellulolytic enhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ ID NO: 65 or SEQID NO: 66) and [FW]-[TF]-K-[AIV], wherein X is any amino acid, X(4,5) isany amino acid at 4 or 5 contiguous positions, and X(4) is any aminoacid at 4 contiguous positions; and (b) a polypeptide havingcellulolytic enhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A-[HNQ] (SEQ ID NO: 79 orSEQ ID NO: 80), wherein X is any amino acid, X(4,5) is any amino acid at4 or 5 contiguous positions, and X(3) is any amino acid at 3 contiguouspositions; wherein the polypeptide having cellulolytic enhancingactivity comprising [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ](SEQ ID NO: 65 or SEQ ID NO: 66) and [FW]-[TF]-K-[AIV] optionallyfurther comprises: H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 67 or SEQID NO: 68), [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 69), orH-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 70 or SEQ ID NO: 71) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 72), wherein X isany amino acid, X(1,2) is any amino acid at 1 position or 2 contiguouspositions, X(3) is any amino acid at 3 contiguous positions, and X(2) isany amino acid at 2 contiguous positions.

[23] The method of paragraph 21, wherein the GH61 polypeptide havingcellulolytic enhancing activity is selected from the group consistingof: (a) a polypeptide comprising or consisting of an amino acid sequencehaving at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, or at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46,SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leastlow stringency conditions, e.g., at least medium stringency conditions,at least medium-high stringency conditions, at least high stringencyconditions, or at least very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ IDNO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, orSEQ ID NO: 63, (ii) the genomic DNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ IDNO: 15, or the cDNA sequence of the mature polypeptide coding sequenceof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, or(iii) a full-length complement of (i) or (ii); (c) a polypeptide encodedby a polynucleotide comprising or consisting of a nucleotide sequencehaving at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ IDNO: 63, or the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 15,or the cDNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ IDNO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO:47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63; (d) a variant ofthe mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ IDNO: 64 comprising a substitution, deletion, and/or insertion of one ormore positions; and (e) a polypeptide having cellulolytic enhancingactivity comprising or consisting of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ IDNO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64; or a fragment thereof havingcellulolytic enhancing activity.

[24] The method of any of paragraphs 21-23, wherein the cellulosicmaterial is pretreated.

[25] The method of paragraphs 21-24, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a hemicellulase, an expansin, an esterase, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[26] The method of paragraph 25, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[27] The method of paragraph 25, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[28] The method of any of paragraphs of 21-27, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, analkane, a cycloalkane, an alkene, isoprene, polyketide, or a gas.

[29] The method of any of paragraphs of 21-28, further comprisingsupplementing the concentration of the divalent copper cation tomaintain the concentration of the divalent copper cation at about 0.0001mM to about 20 mM, e.g., about 0.0005 mM to about 15 mM, about 0.001 mMto about 10 mM, about 0.005 mM to about 5 mM, about 0.01 mM to about 2.5mM, about 0.05 mM to about 1 mM, or about 0.1 mM to about 1 mM.

[30] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more (e.g. several)fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition comprising a GH61 polypeptidehaving cellulolytic enhancing activity and a divalent copper cation,wherein the divalent copper cation is present at a concentration ofabout 0.0001 mM to about 20 mM, e.g., about 0.0005 mM to about 15 mM,about 0.001 mM to about 10 mM, about 0.005 mM to about 5 mM, about 0.01mM to about 2.5 mM, about 0.05 mM to about 1 mM, or about 0.1 mM toabout 1 mM.

[31] The method of paragraph 30, wherein the fermenting of thecellulosic material produces a fermentation product.

[32] The method of paragraph 31, further comprising recovering thefermentation product from the fermentation.

[33] The method of any of paragraphs 30-32, wherein the cellulosicmaterial is pretreated before saccharification.

[34] The method of any of paragraphs 30-33, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[35] The method of paragraph 34, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[36] The method of paragraph 34, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[37] The method of any of paragraphs 31-36, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, analkane, a cycloalkane, an alkene, isoprene, polyketide, or a gas.

[38] The method of any of paragraphs of 30-37, further comprisingsupplementing the concentration of the divalent copper cation tomaintain the concentration of the divalent copper cation at about 0.0001mM to about 20 mM, e.g., about 0.0005 mM to about 15 mM, about 0.001 mMto about 10 mM, about 0.005 mM to about 5 mM, about 0.01 mM to about 2.5mM, about 0.05 mM to about 1 mM, or about 0.1 mM to about 1 mM.

[39] The method of any of paragraphs 10-38, further comprising adding achelator during the degradation or saccharification of the cellulosicmaterial.

[40] The method of paragraph 39, wherein the chelator is selected fromthe group consisting of EDTA (ethylenediaminetetraacetic acid), EGTA(ethyleneglycol-bis-(2-aminoethyl)-N,N,N′,N′-tetraacetic acid), DDTA(3,6-dioxaoctamethylenedinitrilotetraacetic acid), EDDS(ethylenediamine-N,N′-disuccinic acid), BAPTA(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), and BIPY(2,2′-bipyridine).

[41] A composition comprising a GH61 polypeptide having cellulolyticenhancing activity and a divalent copper cation, wherein the divalentcopper cation is present at a concentration of about 0.0001 mM to about20 mM, e.g., about 0.0005 mM to about 15 mM, about 0.001 mM to about 10mM, about 0.005 mM to about 5 mM, about 0.01 mM to about 2.5 mM, about0.05 mM to about 1 mM, or about 0.1 mM to about 1 mM during degradationor saccharification of a cellulosic material and the presence of thedivalent copper cation and the GH61 polypeptide increases thedegradation or conversion of the cellulosic material by an enzymecomposition compared to the GH61 polypeptide without the divalent coppercation.

[42] The composition of paragraph 41, wherein the GH61 polypeptidehaving cellulolytic enhancing activity is selected from the groupconsisting of: (a) a polypeptide having cellulolytic enhancing activitycomprising [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ IDNO: 65 or SEQ ID NO: 66) and [FW]-[TF]-K-[AIV], wherein X is any aminoacid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(4)is any amino acid at 4 contiguous positions; and (b) a polypeptidehaving cellulolytic enhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A-[HNQ] (SEQ ID NO: 79 orSEQ ID NO: 80), wherein X is any amino acid, X(4,5) is any amino acid at4 or 5 contiguous positions, and X(3) is any amino acid at 3 contiguouspositions; wherein the polypeptide having cellulolytic enhancingactivity comprising [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ](SEQ ID NO: 65 or SEQ ID NO: 66) and [FW]-[TF]-K-[AIV] optionallyfurther comprises: H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 67 or SEQID NO: 68), [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 69), orH-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 70 or SEQ ID NO: 71) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 72), wherein X isany amino acid, X(1,2) is any amino acid at 1 position or 2 contiguouspositions, X(3) is any amino acid at 3 contiguous positions, and X(2) isany amino acid at 2 contiguous positions.

[43] The composition of paragraph 41, wherein the GH61 polypeptidehaving cellulolytic enhancing activity is selected from the groupconsisting of: (a) a polypeptide comprising or consisting of an aminoacid sequence having at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, or at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ IDNO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO:64; (b) a polypeptide encoded by a polynucleotide that hybridizes underat least low stringency conditions, e.g., at least medium stringencyconditions, at least medium-high stringency conditions, at least highstringency conditions, or at least very high stringency conditions with(i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO:51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ IDNO: 61, or SEQ ID NO: 63, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, or SEQ ID NO: 15, or the cDNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQID NO: 63, or (iii) a full-length complement of (i) or (ii); (c) apolypeptide encoded by a polynucleotide comprising or consisting of anucleotide sequence having at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, or SEQ ID NO: 63, or the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, or SEQ ID NO: 15, or the cDNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQID NO: 63; (d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ IDNO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,SEQ ID NO: 62, or SEQ ID NO: 64 comprising a substitution, deletion,and/or insertion at one or more positions; and (e) a polypeptide havingcellulolytic enhancing activity comprising or consisting of the maturepolypeptide of of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ IDNO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64;or a fragment thereof having cellulolytic enhancing activity.

[44] The composition of any of paragraphs 41-43, wherein the compositionfurther comprises one or more (e.g., several) enzymes selected from thegroup consisting of a cellulase, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[45] The composition of paragraph 44, wherein the cellulase is one ormore enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[46] The composition of paragraph 44, wherein the hemicellulase is oneor more enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[47] A whole broth formulation or cell culture composition comprising aGH61 polypeptide having cellulolytic enhancing activity and a divalentcopper cation, wherein the divalent copper cation is present at aconcentration of about 0.0001 mM to about 20 mM, e.g., about 0.0005 mMto about 15 mM, about 0.001 mM to about 10 mM, about 0.005 mM to about 5mM, about 0.01 mM to about 2.5 mM, about 0.05 mM to about 1 mM, or about0.1 mM to about 1 mM during degradation or saccharification of acellulosic material and the presence of the divalent copper cation andthe GH61 polypeptide increases the degradation or conversion of thecellulosic material by an enzyme composition compared to the GH61polypeptide without the divalent copper cation.

[48] The whole broth formulation or cell culture composition ofparagraph 47, wherein the GH61 polypeptide having cellulolytic enhancingactivity is selected from the group consisting of: (a) a polypeptidehaving cellulolytic enhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ ID NO: 65 or SEQID NO: 66) and [FW]-[TF]-K-[AIV], wherein X is any amino acid, X(4,5) isany amino acid at 4 or 5 contiguous positions, and X(4) is any aminoacid at 4 contiguous positions; and (b) a polypeptide havingcellulolytic enhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A-[HNQ] (SEQ ID NO: 79 orSEQ ID NO: 80), wherein X is any amino acid, X(4,5) is any amino acid at4 or 5 contiguous positions, and X(3) is any amino acid at 3 contiguouspositions; wherein the polypeptide having cellulolytic enhancingactivity comprising [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ](SEQ ID NO: 65 or SEQ ID NO: 66) and [FW]-[TF]-K-[AIV] optionallyfurther comprises: H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 67 or SEQID NO: 68), [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 69), orH-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 70 or SEQ ID NO: 71) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 72), wherein X isany amino acid, X(1,2) is any amino acid at 1 position or 2 contiguouspositions, X(3) is any amino acid at 3 contiguous positions, and X(2) isany amino acid at 2 contiguous positions.

[49] The whole broth formulation or cell culture composition ofparagraph 47, wherein the GH61 polypeptide having cellulolytic enhancingactivity is selected from the group consisting of: (a) a polypeptidecomprising or consisting of an amino acid sequence having at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, or at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ IDNO: 60, SEQ ID NO: 62, or SEQ ID NO: 64; (b) a polypeptide encoded by apolynucleotide that hybridizes under at least low stringency conditions,e.g., at least medium stringency conditions, at least medium-highstringency conditions, at least high stringency conditions, or at leastvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 15, or the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, or (iii) a full-lengthcomplement of (i) or (ii); (c) a polypeptide encoded by a polynucleotidecomprising or consisting of a nucleotide sequence having at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, or the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 15, or the cDNA sequence of themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO:41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ IDNO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQID NO: 61, or SEQ ID NO: 63; (d) a variant of the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ IDNO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64 comprising asubstitution, deletion, and/or insertion at one or more positions; and(e) a polypeptide having cellulolytic enhancing activity comprising orconsisting of the mature polypeptide of of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:62, or SEQ ID NO: 64; or a fragment thereof having cellulolyticenhancing activity.

[50] The whole broth formulation or cell culture composition of any ofparagraphs 47-49, wherein the composition further comprises one or more(e.g., several) enzymes selected from the group consisting of acellulase, a hemicellulase, an expansin, an esterase, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[51] The composition of paragraph 50, wherein the cellulase is one ormore enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[52] The composition of paragraph 50, wherein the hemicellulase is oneor more enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A composition comprising a GH61 polypeptidehaving cellulolytic enhancing activity and a divalent copper cation,wherein the divalent copper cation is present at a concentration ofabout 0.0001 mM to about 20 mM during degradation or saccharification ofa cellulosic material and the presence of the divalent copper cation andthe GH61 polypeptide increases the degradation or conversion of thecellulosic material by the composition compared to the GH61 polypeptidewithout the divalent copper cation.
 2. The composition of claim 1, whichfurther comprises one or more enzymes selected from the group consistingof a cellulase, a hemicellulase, an expansin, an esterase, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.
 3. The composition of claim 2, wherein the cellulase is oneor more enzymes selected from the group consisting of an endoglucanase,a cellobiohydrolase, and a beta-glucosidase.
 4. The composition of claim2, wherein the hemicellulase is one or more enzymes selected from thegroup consisting of a xylanase, an acetylxylan esterase, a feruloylesterase, an arabinofuranosidase, a xylosidase, and a glucuronidase. 5.A whole broth formulation or cell culture composition comprising a GH61polypeptide having cellulolytic enhancing activity and a divalent coppercation, wherein the divalent copper cation is present at a concentrationof about 0.0001 mM to about 20 mM during degradation or saccharificationof a cellulosic material and the presence of the divalent copper cationand the GH61 polypeptide increases the degradation or conversion of thecellulosic material by the formulation or the composition compared tothe GH61 polypeptide without the divalent copper cation.
 6. The wholebroth formulation or cell culture composition of claim 5, which furthercomprises one or more enzymes selected from the group consisting of acellulase, a hemicellulase, an expansin, an esterase, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.
 7. The whole broth formulation or cell culture composition ofclaim 6, wherein the cellulase is one or more enzymes selected from thegroup consisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase.
 8. The whole broth formulation or cell culturecomposition of claim 6, wherein the hemicellulase is one or more enzymesselected from the group consisting of a xylanase, an acetylxylanesterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, anda glucuronidase.